Medicaments against viral diseases

ABSTRACT

Novel dihydropyrimidines and combinations thereof with other antiviral agents, suitable for combating HBV infections.

The present invention relates to novel 6-aminoalkyldihydropyrimidines,processes for their preparation and their use as medicaments, inparticular for the treatment and prophylaxis of hepatitis B virusinfections. The invention also relates to combinations of thesedihydropyrimidines with other antiviral agents and, where appropriate,immunomodulators, and to medicaments comprising these combinations, inparticular for the treatment and prophylaxis of HBV infections such ashepatitis B.

The hepatitis B virus belongs to the family of hepadna viruses. Itcauses an acute and/or a persistent/progressive chronic disease. Manyother clinical manifestations in the pathological state are also causedby the hepatitis B virus—in particular chronic inflammation of theliver, cirrhosis of the liver and hepatocellular carcinoma. In addition,coinfection with the heptatitis delta virus may have adverse effects onthe progress of the disease.

The only agents approved for the treatment of chronic hepatitis areinterferon and lamivudine. However, interferon has only moderateactivity and has unwanted side effects; although lamivudine has goodactivity, resistance develops rapidly during treatment and a reboundeffect occurs in most cases after discontinuation of the therapy.

EP-B 103 796 discloses dihydropyrimidines attributed with an effectinfluencing the circulation. WO 99/1438 relates to dihydropyrimidinessaid to be suitable for the treatment of cerebrovascular ischemia and ofpain. WO 99/54312, 99/54326 and 99/54329 relate to dihydropyrimidinessuitable for the treatment and prophylaxis of hepatitis.

The present invention relates to compounds of the formula

and the isomeric form thereof

in which

-   R¹, R² are, independently of one another, hydrogen, fluorine,    chlorine or bromine,-   R³ is C₁–C₄-alkyl,-   X is a methylene or ethylene group,-   Z is NR⁴R⁵ or pyridyl,-   R⁴ is C₁–C₄-alkyl which may be substituted by hydroxyl or    C₁–C₄-alkoxycarbonyl, or is benzyl,-   R⁵ is C₁–C₄-alkyl which may be substituted by hydroxyl,    or-   R⁴ and R⁴ together with the nitrogen atom to which they are bonded    are an imidazolyl, triazolyl or tetrazolyl ring or a radical of the    formula

in which

-   -   a is zero or 1 and    -   Y is CH₂, CH₂CH₂, —O— or —S—,    -   and    -   R⁶ is pyridyl which is substituted once to twice by fluorine, or        is thiazolyl,    -   and the salts thereof.

Preference is given to compounds of the invention of the formulae (I)and (Ia) in which

-   R⁴ and R⁵ together with the nitrogen atom to which they are bonded    form a morpholinyl or thiomorpholinyl ring,    and the salts thereof.

Particular preference is given to compounds of the invention of theformulae (I) and (Ia) in which

-   R¹, R² are, independently of one another, fluorine, chlorine or    bromine,    and the salts thereof.

Very particular preference is given to compounds of the invention of theformulae (I) and (Ia) in which

-   R¹, R² are, independently of one another, fluorine, chlorine or    bromine, and-   R⁴ and R⁵ together with the nitrogen atom to which they are bonded    form a morpholinyl or thiomorpholinyl ring,    and the salts thereof.

Alkyl is within the framework of the invention a linear or branchedalkyl radical having 1 to 4 carbon atoms, such as, for example, methyl,ethyl, propyl, isopropyl and tert-butyl.

Alkoxycarbonyl is within the framework of the invention a linear orbranched alkoxycarbonyl radical having 1 to 4 carbon atoms, such as, forexample, methoxycarbonyl, ethoxycarbonyl and propoxycarbonyl.

The compounds of the invention may exist in stereoisomeric forms whicheither are related as image and mirror image (enantiomers) or are notrelated as image and mirror image (diastereomers). The invention relatesboth to the enantiomers or diastereomers and to the respective mixturesthereof. The racemic forms can, just like the diastereomers, beseparated in a manner known per se into the stereoisomerically uniformcomponents.

The compounds of the invention include the isomers of the formulae (I)and (Ia) and the mixtures thereof. The compounds of the invention mayalso be in the form of salts. Physiologically acceptable salts arepreferred within the framework of the invention.

Physiologically acceptable salts may be salts of inorganic or organicacids. Preference is given to salts of inorganic acids such as, forexample, hydrochloric acid, hydrobromic acid, phosphoric acid orsulfuric acid, or salts of organic carboxylic or sulfonic acids such as,for example, acetic acid, maleic acid, fumaric acid, malic acid, citricacid, tartaric acid, lactic acid, benzoic acid or methanesulfonic acid,ethanesulfonic acid, phenylsulfonic acid, toluenesulfonic acid ornaphthalene-disulfonic acid.

Physiologically acceptable salts may also be metal or ammonium salts ofthe compounds of the invention. Particularly preferred examples aresodium, potassium, magnesium or calcium salts, and ammonium saltsderived from ammonia or organic amines such as, for example, ethylamine,di- or triethylamine, di- or triethanolamine, dicyclohexylamine,dimethylaminoethanol, arginine, lysine, ethylenediamine or2-phenylethylamine.

The compounds (I) of the invention can be prepared by

-   [A] firstly converting aldehydes of the formula

in which R¹ and R² have the meanings indicated above,with β-ketoesters of the formula

in which R³, X and Z have the meanings indicated above,with or without addition of base or acid, where appropriate in thepresence of inert organic solvents, into benzylidene compounds of theformula

and then reacting the latter with amidines of the formula

in which

-   R⁶ has the meaning indicated above,    or the salts thereof (such as, for example, hydrochlorides or    acetates) with or without addition of base or acid, where    appropriate in the presence of inert organic solvents, or-   [B] reacting compounds of the formula (III) in a one-stage process    with aldehydes (II) and amidines (V) or salts thereof (such as, for    example, hydrochlorides or acetates) with or without addition of    base or acid, where appropriate in the presence of inert organic    solvents, or else-   [C] where X in formula (I) is a methylene group, reacting compounds    of the formula

in which

-   R¹, R², R³ and R⁶ have the meanings indicated above, and-   Y is a nucleophilically replaceable group such as chloride, bromide,    iodide, mesylate or tosylate,    with compounds of the formula

in which

-   R⁴ and R⁵ have the meanings indicated above,    with or without addition of a base, where appropriate in inert    solvents.

The compounds (VI) can be prepared, for example, by converting compoundsof the formula

in which

-   R¹, R², R³ and R⁶ have the meanings indicated above,    with a brominating agent such as, for example, N-bromosuccinimide,    preferably in the presence of inert solvents, into compounds of the    formula

The latter can then be reacted, directly or after furthertransformation, customary in the literature, of the nucleophilicallyreplaceable group, with compounds (VII).

-   [D] Where X, in formula (I) is an ethylene group, it is also    possible to convert compounds of the formula

in which

-   R¹, R² and R³ have the meanings indicated above,    with immonium salts of the formula

in which

-   R⁴ and R⁴ have the meanings indicated above,    with or without addition of a base, where appropriate in inert    solvents, into compounds of the formula

and then to react the latter with amidines (V) or salts thereof (suchas, for example, hydrochlorides or acetates) with or without addition ofacid or base, where appropriate in the presence of inert organicsolvents.

To prepare the compounds of the invention of the formula (I) in which Xis a methylene group and Z is the group —NR⁴R⁵, the corresponding β-ketocarboxylic esters (III) can also be obtained by reacting chloroaceticesters of the formula

in which

-   R³ has the meaning indicated above,    with compounds of the formula (VII).

To prepare the compounds of the invention of the formula (I) in which Xis an ethylene group and Z is pyridyl, the corresponding β-ketocarboxylic esters (III) can also be obtained by reacting the dianions ofacetoacetic esters of the formula

in which

-   R³ has the meaning indicated above,    with picolyl derivatives of the formula

in which

-   Y has the meaning indicated above.

The aldehydes (II) used as starting materials are known or can beprepared by methods known from the literature [cf. T. D. Harris and G.P. Roth, J. Org. Chem. 44, 146 (1979); DE-A 2 165 260 and 2 401 665;Mijano et al., Chem. Abstr. 59, 13 929 c (1963); E. Adler and H.-D.Becker, Chem. Scand. 15, 849 (1961); E. P. Papadopoulos, M. Mardin andCh. Issidoridis, J. Org. Chem. Soc. 78, 2543, (1956)].

The β-keto carboxylic esters (III) used as starting materials are knownin some cases or can be prepared in analogy to methods known from theliterature [e.g. D. Borrmann, “Umsetzung von Diketen mit Alkoholen,Phenolen und Mercaptanen”, in “Methoden der Organischen Chemie”(Houben-Weyl), vol. VII/4, 230 ff (1968); Y. Oikawa, K. Sugano und O.Yonemitsu, J. Org. Chem. 43, 2087 (1978)].

The compounds (V) are known in some cases or can be prepared asdescribed in WO-A-99/54326 and WO-A-99/54329.

The compounds (VIII) and (X) can be prepared in accordance with processvariants [A] or [B] as described in WO-A-99/54326.

The compounds (VII) and (XI) are known or can be prepared by customarymethods.

Solvents suitable for all the process variants A, B, C, D and E are allinert organic solvents. These preferably include alcohols such asmethanol, ethanol, isopropanol, ethers such as dioxane, diethyl ether,tetrahydrofuran, glycol monomethyl ether, glycol dimethyl ether,carboxylic acids such as glacial acetic acid, or dimethylformamide,dimethyl sulfoxide, acetonitrile, pyridine and hexamethylphosphorictriamine.

The reaction temperatures may be varied within a relatively wide range.Those used are generally between 20 and 150° C., but preferably at theboiling point of the particular solvent.

The reaction can be carried out under atmospheric pressure but alsounder elevated pressure. It is generally carried out under atmosphericpressure.

The reaction can be carried out with or without addition of base oracid; however, it is advisable to carry out the reaction in the presenceof relatively weak acids such as, for example, acetic acid or formicacid.

Compounds of the formula (IX) are novel; the invention therefore alsorelates to compounds of the formula (IX).

One embodiment of the invention relates to combinations of A) at leastone of the dihydropyrimidines defined above, B) at least one otherantiviral agent different from A.

A particular embodiment of the invention relates to combinations of A)above dihydropyrimidines, B) HBV polymerase inhibitors and, whereappropriate, C) immunomodulators.

Preferred immunomodulators C) comprise, for example, all interferonssuch as α-, β- and γ-interferons, in particular also α-2a- andα-2b-interferons, interleukins such as interleukin-2, polypeptides suchas thymosin-α-1 and thymoctonan, imidazoquinoline derivatives such as®Levamisole, immunoglobulins and therapeutic vaccines.

The invention thus also relates to these combinations for the treatmentand prophylaxis of HBV infections and to the use thereof for thetreatment of HBV-induced diseases.

The use of the combinations of the invention provides valuableadvantages for the treatment of HBV-induced diseases compared withmonotherapy with the individual compounds, namely principally asynergistic antiviral activity, but also good tolerability of thecombinations of the invention in the range of toxicity at which 50% ofthe cells survive (“Tox-50”)—compared with the Tox-50 of the individualcomponents.

The substances referred to as HBV polymerase inhibitors B for thepurposes of the invention are those which, in the endogenous polymeraseassay which was published by Ph. A. Furman et al. in AntimicrobialAgents and Chemotherapy, Vol. 36 (No. 12), 2688 (1992) and which isdescribed hereinafter, lead to an inhibition of the formation of an HBVDNA double strand, so as to result in a maximum of 50% of the activityof the zero value:

HBV virions from culture supernatants incorporate nucleoside5′-triphosphates into the plus strand of the HBV DNA in vitro. By usingagarose gel electrophoresis, the incorporation of[α-³²P]-deoxynucleoside 5′-triphosphate into the viral 3.2 kb DNAproduct is observed in the presence and absence of a substancepotentially having HBV polymerase-inhibiting properties. HBV virions areobtained from the cell culture supernatant of HepG2.2.15 cells byprecipitation with polyethylene glycol and are concentrated. 1 part byvolume of clarified cell culture supernatant is mixed with ¼ by volumeof an aqueous solution containing 50% by weight polyethylene glycol 8000and 0.6 M sodium chloride. The virions are sedimented by centrifugationat 2500×g/15 minutes. The sediments are resuspended in 2 ml of buffercontaining 0.05 M tris-HCl (pH 7.5) and dialyzed against the same buffercontaining 100 mM potassium chloride. The samples can be frozen at −80°C. Each reaction mixture (100 μl) contains at least 10⁵ HBV virions; 50mM tris-HCl (p_(H) 7.5); 300 mM potassium chloride; 50 mM magnesiumchloride; 0.1% ®Nonident P-40 (nonionic detergent from BoehringerMannheim); 10 μM each dATP, dGTP and dTTP; 10 μCi of [³²P]dCTP (3000Ci/mmol; final concentration 33 nM) and 1 μM of the potential polymeraseinhibitor in its triphosphorylated form. The samples are incubated at37° C. for one hour and then the reaction is stopped by adding 50 mMEDTA. A 10% weight/volume SDS solution (containing 10 g of SDS per 90 mlof water) is added to a final concentration of 1% by volume (based onthe total volume), and proteinase K is added to a final concentration of1 mg/ml. After incubation at 37° C. for one hour, samples are extractedwith the same volume of phenol/chloroform/isoamyl alcohol (ratio 25:24:1by volume), and the DNA is precipitated from the aqueous phase withethanol. The DNA pellet is resuspended in 10 μl of gel buffer (solutionof 10.8 g of tris, 5.5 g of boric acid and 0.75 g of EDTA in 1 liter ofwater (=TBE buffer)) and separated by electrophoresis in an agarose gel.Either the gel is dried or the nucleic acids present therein transferredby the Southern transfer technique to a membrane. The amount of labeledDNA double strand formed is then determined in relation to the negativecontrol (=endo-pol reaction without substance or with inactive controlsubstance). An HBV polymerase inhibitor is present if a maximum of 50%of the activity of the negative control is present.

Preferred HBV polymerase inhibitors B) comprise, for example,

-   3TC=lamivudine==4-amino-1-[(2R-cis)-2-(hydroxymethyl)-1.3-oxathiolan-5-yl]-pyrimidin-2(1H)-one,    cf. EP-B 382 526 (=U.S. Pat. No. 5,047,407) and WO 91/11186 (=U.S.    Pat. No. 5,204,466);-   Adefovir    dipivoxil=9-{2-[[bis[(pivaloyloxy)-methoxy]-phosphinyl]-methoxy]-ethyl}-adenine,    cf. EP-B 481 214 (=U.S. Pat. Nos. 5,663,159 and 5,792,756), U.S.    Pat. Nos. 4,724,233 and 4,808,716;-   BMS 200    475=[1S-(1.α,3.α,4.β)]-2-amino-1.9-dihydro-9-[4-hydroxy-3-(hydroxymethyl)-2-methylene-cyclopentyl]-6H-purin-6-one,    cf. EP-B 481 754 (=U.S. Pat. Nos. 5,206,244 and 5,340,816), WO    98/09964 and 99/41275;-   Abacavir=(−)-(1S-cis)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol,    cf. EP-B 349 242 (=U.S. Pat. No. 5,049,671) and EP-B 434 450 (=U.S.    Pat. No. 5,034,394);-   FTC=(2R-cis)-4-amino-5-fluoro-1-[2-(hydroxymethyl)-1.3-oxathiolan-5-yl]-pyrimidin-2(1H)-one,    cf. WO 92/14743 (=U.S. Pat. Nos. 5,204,466, 5,210,085, 5,539,116,    5,700,937, 5,728,575, 5,814,639, 5,827,727, 5,852,027, 5,892,025,    5,914,331, 5,914,400) and WO 92/18517;-   β-L-FDDC=5-(6-amino-2-fluoro-9H-purin-9-yl)-tetrahydro-2-furanmethanol,    cf. WO 94/27616 (=U.S. Pat. Nos. 5,627,160, 5,561,120, 5,631,239 and    5,830,881);-   L-FMAU=1-(2-deoxy-2-fluoro-β-L-arabinofuranosyl)-5-methyl-pyrimidine-2.4(1H,3H)-dione,    cf. WO 99/05157, WO 99/05158 and U.S. Pat. No. 5,753,789.

A further preferred embodiment of the invention relates to combinationsof A) above dihydropyrimidines (I) and (Ia) and B) lamivudine.

Other preferred HBV antiviral agents B comprise, for example,phenylpropenamides of the formula

in which

-   R¹ and R² are, independently of one another, C₁–₄-alkyl or, together    with the nitrogen atom on which they are located, form a ring having    5 to 6 ring atoms which comprise carbon and/or oxygen,-   R³ to R¹² are, independently of one another, hydrogen, halogen,    C₁–₄-alkyl, optionally substituted C₁–₄-alkoxy, nitro, cyano or    trifluoromethyl,-   R¹³ is hydrogen, C₁–₄-alkyl, C₁–₇-acyl or aralkyl and-   X is halogen or optionally substituted C₁–₄-alkyl,    and the salts thereof.

These phenylpropenamides and processes for preparing them are disclosedin WO 98/33501, to which reference is hereby made for the purpose of thedisclosure. AT-61 is the compound of the above formula in which X ischlorine, A is 1-piperidinyl and Y and Z are each phenyl.

Preferred immunomodulators C) comprise, for example, all interferonssuch as α-, β- and γ-interferons, in particular also α-2a- andα-2b-interferons, interleukins such as interleukin-2, polypeptides suchas thymosin-α-1 and thymoctonan, imidazoquinoline derivatives such as®Levamisole, immunoglobulins and therapeutic vaccines.

A further preferred embodiment of the invention relates to combinationsof A) above dihydropyrimidines (I) and (Ia), B) lamivudine and, whereappropriate, C) interferon.

Description of Tests

The antiviral action of the compounds of the invention on hepatitis Bvirus was investigated by methods based on those described by M. A.Sells et al., Proc. Natl. Acad. Sci. 84, 1005–1009 (1987) and B. E.Korba et al., Antiviral Research 19, 55–70 (1992).

The antiviral tests were carried out in 96-well microtiter plates. Thefirst vertical row of the plate received only growth medium andHepG2.2.15 cells. It served as virus control.

Stock solutions of the test compounds (50 mM) were initially dissolvedin DMSO, and further dilutions were prepared in the HepG2.2.15 growthmedium. The compounds according to the invention were usually pipettedin a test concentration of 100 μM (1st test concentration) in each caseinto the second vertical test row of the microtiter plate andsubsequently diluted in twofold steps 2¹⁰ times in growth medium plus 2%by weight of fetal calf serum (volume 25 μl).

Each well of the microtiter plate then contained 225 μl of HepG2.2.15cell suspension (5×10⁴ cells/ml) in growth medium plus 2% by weight offetal calf serum. The test mixture was incubated at 37° C. and 5% CO₂(v/v) for 4 days.

The supernatant was then aspirated off and discarded, and the wellsreceived 225 μl of freshly prepared growth medium. The compoundsaccording to the invention were each added anew as 10-fold concentratedsolution in a volume of 25 μl. The mixtures were incubated for a further4 days.

Before harvesting the supernatants to determine the antiviral effect,the HepG2.2.15 cells were examined under the light microscope or bymeans of biochemical detection methods (for example Alamar Blue stain orTrypan Blue stain) for cytotoxic changes.

The supernatant and/or cells were then harvested and sucked by means ofa vacuum onto 96-well dot-blot chambers covered with a nylon membrane(in accordance with the manufacturer's information).

Cytotoxicity Determination

Substance-induced cytotoxic or cytostatic changes in the HepG2.2.15cells were detected, for example, under the light microscope as changesin cell morphology. Such substance-induced changes in the HepG2.2.15cells compared with untreated cells were visible, for example, ascytolysis, vacuolation or altered cell morphology. 50% cytotoxicity(Tox.-50) means that 50% of the cells show a morphology comparable tothe corresponding cell control.

The tolerability of some of the compounds according to the invention wasadditionally tested on other host cells such as, for example, HeLacells, primary human peripheral blood cells or transformed cell linessuch as H-9 cells.

No cytotoxic changes were detectable at concentrations >10 μM of thecompounds of the invention.

Determination of the Antiviral Action

After the supernatants or lysed cells had been transferred to the nylonmembrane of the blot apparatus (see above), the intra- or extracellularsupernatants of the HepG2.2.15 cells were denatured (1.5 M NaCl/0.5 NNaOH), neutralized (3 M NaCl/0.5 M Tris HCl, pH 7.5) and washed (2×SSC).The DNA was then baked onto the membrane by incubating the filters at120° C. for 2–4 hours.

DNA Hybridization

Detection of the viral DNA from the treated HepG2.2.15 cells on thenylon filters was usually carried out with nonradioactive,digoxigenin-labeled hepatitis B-specific DNA probes, each of which waslabeled with digoxigenin, purified and employed for the hybridization inaccordance with the manufacturer's information.

The prehybridization and hybridization took place in 5×SSC, 1× blockingreagent, 0.1% by weight N-lauroylsarcosine, 0.02% by weight SDS and 100μg of herring sperm DNA. The prehybridization took place at 60° C. for30 minutes, and the specific hybridization with 20 to 40 ng/ml of thedigoxigenized, denatured HBV-specific DNA (14 hours, 60° C.). Thefilters were then washed.

Detection of HBV-DNA by Digoxigenin Antibodies

The immunological detection of the digoxigenin-labeled DNA took place inaccordance with the manufacturer's information:

The filters were washed and prehybridized in a blocking reagent (inaccordance with the manufacturer's information). Hybridization was thencarried out with an anti-DIG antibody coupled to alkaline phosphatasefor 30 minutes. After a washing step, the substrate of alkalinephosphatase, CSPD, was added, incubated with the filters for 5 minutes,then packed in plastic film and incubated at 37° C. for a further 15minutes. The chemiluminescence of the hepatitis B-specific DNA signalswas visualized by exposing the filters to an X-ray film (incubationdepending on signal strength: 10 minutes to 2 hours).

The half-maximum inhibitory concentration (IC₅₀, 50% inhibitoryconcentration) was determined as the concentration at which the intra-or extracellular hepatitis B-specific band was reduced by the compoundaccording to the invention by 50% compared with an untreated sample.

The compounds of the invention show a valuable antiviral action whichcould not have been predicted. They surprisingly show antiviral activityagainst hepatitis B viruses (HBV) since they cause an extremely largereduction in intra- and/or extracellular HBV DNA. The compounds of theinvention are thus suitable for the treatment of virus-induced diseases,in particular of acutely and chronically persistent viral infections byHBV. A chronic viral disease caused by HBV may lead to pathologicalstates varying in severity, and chronic hepatitis B viral infection isknown in many cases to lead to cirrhosis of the liver and/orhepatocellular carcinoma.

Areas of indication which may be mentioned for the compounds of theinvention are, for example:

-   the treatment of acute and chronic viral infections which may lead    to infectious hepatitis, for example infections with heptatitis B    viruses. The compounds of the invention are particularly suitable    for the treatment of chronic hepatitis B infections and the    treatment of acute and chronic hepatitis B viral infections.

The present invention includes pharmaceutical preparations which,besides nontoxic, inert pharmaceutically suitable carriers, comprise oneor more compounds (I) or (Ia) or a combination of the invention or whichconsist of one or more active ingredients (I) or (Ia) or of acombination of the invention.

The active ingredients (I) and (Ia) are intended to be present in thepharmaceutical preparations mentioned above in a concentration of about0.1 to 99.5% by weight, preferably of about 0.5 to 95% by weight, of thecomplete mixture.

The pharmaceutical preparations mentioned above may also comprise otheractive pharmaceutical ingredients apart from the compounds (I) and (Ia).

The ratio of the amounts of the components A, B and, where appropriate,C in the combinations of the invention may vary within wide limits; itis preferably 5 to 500 mg of A/10 to 1000 mg of B, in particular 10 to200 mg of A/20 to 400 mg of B.

Component C, which is also to be used where appropriate, may be used inamounts of, preferably, 1 to 10 million, in particular 2 to 7 million,I.U. (international units), about three times a week over a period of upto one year.

The compounds or combinations of the invention are intended to bepresent in the pharmaceutical preparations mentioned above in general ina concentration of about 0.1 to 99.5, preferably about 0.5 to 95, % byweight of the complete mixture.

The pharmaceutical preparations mentioned above can be produced in aconventional way by known methods, for example by mixing the activeingredient(s) with the carrier(s).

It has generally proved to be advantageous both in human and inveterinary medicine to administer the active ingredient(s) in totalamounts of about 0.5 to about 500, preferably of 1 to 100 mg/kg of bodyweight every 24 hours, where appropriate in the form of a plurality ofsingle doses, to achieve the desired results. A single dose contains theactive ingredient(s) preferably in amounts of about 1 to about 80, inparticular 1 to 30, mg/kg of body weight. However, it may be necessaryto deviate from the dosages mentioned, in particular depending on thespecies and body weight of the subject to be treated, the nature andseverity of the disorder, the type of preparation and mode ofadministration of the medicament, and the time or interval within whichadministration takes place.

The invention therefore relates further to the compounds andcombinations defined above for controlling diseases.

The invention further relates to medicaments comprising at least one ofthe compounds or combinations defined above and, where appropriate, oneor more other active pharmaceutical ingredient(s).

The invention further relates to the use of the compounds andcombinations defined above for producing a medicament for the treatmentand prophylaxis of the diseases described above, preferably of viraldiseases, in particular of hepatitis B.

The percentage data in the following examples relate in each case toweight unless indicated otherwise. The ratios of solvents in solventmixtures are in each case based on volume.

EXAMPLES A. Starting Compounds Example I 3-Fluoropyridine N-oxide

22.20 ml of H₂O₂ (30% strength) are added to a solution of 11.10 g(114.324 mmol) of 3-fluoropyridine in 74.00 ml of acetic acid, and themixture is stirred at a bath temperature of 100° C. for 7 hours. It isthen concentrated to 30 ml and, after addition of 30 ml of water, againconcentrated to 30 ml. The solution is stirred with dichloromethane,basified by addition of K₂CO₃ and separated, and the aqueous phase isextracted twice with dichloromethane, dried and concentrated.

Yield: 11.5 g (88.9%)

Melting point: 66–68° C.

Example II 2-Cyano-3-fluoropyridine

5.20 g (45.980 mmol) of the compound from Example I are dissolved in 50ml of acetonitrile. Under argon, 13.70 g (138.092 mmol) oftrimethylsilylnitrile are added and 12.80 ml of triethylamine are slowlyrun in. The solution is stirred under reflux for 7 hours and then atroom temperature overnight. Concentration using a waterpump is followedby taking up in dichloromethane, shaking twice with 50 ml of 2N aqueoussodium carbonate solution, washing with water, drying and concentrating.

Yield (crude): 5.3 g (oil)

Column chromatography: methylene chloride to methylene chloride/ethylacetate (10:1)

Example III 2-Amidino-3-fluoropyridine hydrochloride

A sodium methoxide solution made from 0.40 g (17.391 mmol) of sodium and65 ml of methanol is added to a solution of 10.30 g (84.355 mmol) of thecompound from Example II in 30 ml of methanol, and the mixture isstirred at 20° C. for 72 hours. 5.44 g (101.682 mmol) of ammoniumchloride (powdered) and 17.39 mmol (1.04 ml) of acetic acid are added,and the mixture is stirred at 40° C. for 28 hours and cooled. Insolublesalt is filtered off with suction (1.78 g), and the filtrate isconcentrated, concentrated with acetone and then stirred with acetone,filtered off with suction and washed.

Yield: 10.6 g

Melting point: ≈150° C. decomposition

Example IV 2-Cyano-3,5-dichloropyridine

Method 1:

21.8 ml (0.174 mmol) of trimethylsilyl cyanide and 14.6 ml (0.158 mmol)of dimethylcarbamoyl chloride are added successively to a solution of 26g (0.158 mol) of 3,5-dichloropyridine 1-oxide (Johnson et al., J. Chem.Soc. B, 1967, 1211) in 80 ml of dichloromethane and stirred at roomtemperature for 48 hours. 100 ml of a 10% strength aqueous NaHCO₃solution are added, and the mixture is vigorously stirred for 10minutes. Separation of the phases is followed by extraction once withdichloromethane; the combined organic phases are dried and concentrated.The residue is chromatographed on silica gel with dichloromethane andrecrystallized from a little methanol.

11 g (40.2%) of 2-cyano-3,5-dichloropyridine (melting point: 102° C.)are obtained.

Method 2:

In analogy to Troschuetz, R. et al., J. Heterocycl. Chem. 33, 1815–1821(1996), 150 ml of diethylene glycol dimethyl ether, 47.68 g (0.261 mol)of 2,3,5-trichloro-pyridine, 2.0 g (0.005 mol) of tetraphenylphosphoniumbromide, 4.0 g (0.024 mol) of finely powdered potassium iodide and 75.0g (0.838 mol) of copper(I) cyanide are mixed under nitrogen and stirredunder reflux for 24 hours. Then a further 100 ml of diethylene glycoldimethyl ether, 2.0 g (0.005 mol) of tetraphenylphosphonium bromide, 4.0g (0.024 mol) of finely powdered potassium iodide and 75 g (0.838 mol)of copper(I) cyanide are added, and the mixture is stirred at refluxtemperature for a further 89 hours. Cooling to room temperature isfollowed by filtration with suction, and the filtrate is distilled toremove most of the diethylene glycol dimethyl ether. The residue istaken up in toluene and washed with an aqueous solution of Mohr's saltand then with aqueous NaHCO₃ solution (peroxide test). It is then washedwith water to remove diethylene glycol dimethyl ether. After filtrationthrough cellit, the filtrate is dried over magnesium sulfate, and thesolution is concentrated.

18.0 g (40.0%) of 2-cyano-3,5-dichloropyridine are obtained.

Example V 2-Cyano-3,5-difluoropyridine

50 g (0.29 mol) of 2-cyano-3,5-dichloropyridine from Example IV, 33.6 g(0.58 mol) of potassium fluoride and 10 g of polyethylene glycol 8000are mixed with 125 ml of DMSO and heated at 160° C. for 30 minutes.After cooling, the product is distilled out together with the DMSO underhigh vacuum, the distillate is added to water and, after extraction withtoluene, dried over sodium sulfate. The product is reacted further assolution in toluene.

R_(f): 0.43 (cyclohexane/ethyl acetate=7:3)

Example VI 3,5-Difluoro-2-pyridinecarboximidamide hydrochloride

328 ml of trimethylaluminum (2 M in hexane, 0.624 mol) are addeddropwise to a suspension, cooled to 0 to 5° C., of 33.4 g (0.624 mol) ofammonium chloride in 1 l of toluene; the mixture is stirred at roomtemperature until methane evolution has ceased. The solution of2-cyano-3,5-dichloropyridine from Example V in toluene is then addeddropwise, and the mixture is subsequently stirred at 80° C. overnight.After cooling to 0 to −5° C., methanol is added dropwise until gasevolution ceases, and the salts are filtered off with suction and washedtwice with a little methanol. The solvent is stripped off, the residueis dissolved in 500 ml of dichloromethane/methanol (9:1) and againfiltered with suction to remove inorganic salts. 23.6 g (39.1%) of3,5-difluoro-2-pyridinecarboximidamide hydrochloride (melting point:183° C.) remain after the solvent has been stripped off.

¹H-NMR (DMSO-D₆): 8.3–8.45 (m, 1H) ppm; 8.8 (d, J=2 Hz, 1H) ppm; 9.7 (s,broad, 4H) ppm.

Example VII Methyl 2-acetyl-3-(2-chloro-4-fluorophenyl)acrylate

1.7 ml of piperidine acetate are added to a solution of 50 g (315 mmol)of 2-chloro-4-fluorobenzaldehyde and 36.6 g (315 mmol) of methylacetoacetate in 150 ml of isopropanol. Stirring at room temperatureovernight is followed by dilution with dichloromethane and extractionwith water, and the organic phase is dried over sodium sulfate andconcentrated. The product is reacted further as a crude cis/transmixture.

Example VIII Methyl 4-(4-morpholinyl)-3-oxobutanoate

1.27 g (14.6 mmol) of morpholine are added to a solution of 1.0 g (6.64mmol) of methyl 4-chloroacetoacetate in 10 ml of dichloromethane, andthe mixture is stirred at room temperature for 4 hours. Water is thenadded and the mixture is neutralized with 2N hydrochloric acid. Theorganic phase is dried over sodium sulfate and concentrated, and theresidue is chromatographed on silica gel with cyclohexane/ethyl acetate(10:1→4:1→1:1) as mobile phase.

Yield: 0.475 g (35.5)

Example IX Methyl 3-(2-chloro-4-fluorophenyl)-2-[3-(1,3-thiazolidin-3-yl)-propanoyl]-acrylate

3.24 g (12.6 mmol) of methyl2-acetyl-3-(2-chloro-4-fluorophenyl)acrylate from Example VII arestirred with 1.74 g (12.6 mmol) of 3-methylene-1,3-thiazolidin-3-iumchloride [prepared in analogy to H. Mohrle et al., Z. Naturforsch. 42,1035–1046 (1987)] in 50 ml of acetonitrile at 40° C. for 48 hours. Afterconcentration, the residue is taken up in ethyl acetate and washed withwater. The organic phase is dried over sodium sulfate and concentrated,and the residue is chromatographed on silica gel with cyclohexane/ethylacetate (5:1→2:1) as mobile phase.

Yield: 0.67 g (14.8%)

Example X Methyl 3-oxo-5-(4-pyridinyl)-pentanoate

10.0 g (86.1 mmol) of methyl acetoacetate are added dropwise to 3.62 g(90.5 mmol) of sodium hydride (as 60% suspension in mineral oil) in 150ml of THF at 0° C. After stirring at 0° C. for a further 20 minutes, 53ml of a 1.6 molar solution of n-butyl-lithium in hexane are addeddropwise and then, after stirring for 10 minutes, a solution of 14.1 g(86.1 mmol) of 4-picolyl chloride in 65 ml of THF is added. Stirring atroom temperature overnight is followed by neutralization with 2Nhydrochloric acid and separation of the phases, and the organic phase isdried over sodium sulfate and concentrated, and the crude product ispurified by column chromatography on silica gel firstly with petroleumether/ethyl acetate (4:1) and then with dichloromethane/methanol (9:1)as mobile phase.

Yield: 3.2 g (17.9% of theory)

R_(f)=0.62 (dichloromethane/methanol 10:1)

Example XI Methyl3-(2-chloro-4-fluorophenyl)-2-[3-(4-pyridinyl)-propanoyl]-2-propenoate

A solution of 2.44 g (15.4 mmol) of 2-chloro-4-fluorobenzaldehyde and3.19 g (15.4 mmol) of the compound from Example X in 20 ml ofisopropanol are mixed with 0.1 ml of piperidine and 0.13 ml of glacialacetic acid. Stirring at room temperature overnight is followed byconcentration, and the residue is chromatographed on silica gel withdichloromethane->dichloromethane/methanol (50:1) as mobile phase. Theproduct is employed further as cis/trans mixture.

Yield: 4.5 g (84.0% of theory)

Example XII Methyl4-(2-chloro-4-fluorophenyl)-2-(3,5-difluoro-2-pyridinyl)-6-methyl-1,4-dihydropyrimidine-5-carboxylate

4.5 g (23.2 mmol) of 3,5-difluoro-2-pyridinecarboximidamidehydrochloride from Example VI and 7.7 g (30 mmol) of methyl2-acetyl-3-(2-chloro-4-fluorophenyl)-2-propenoate from Example VII and2.3 g (27.9 mmol) of sodium acetate are dissolved or suspended in 120 mlof isopropanol and boiled under reflux for 4 hours. Cooling to roomtemperature is followed by filtration with suction to remove inorganicsalts, and concentration. The residue is taken up in a mixture of 30 mlof 1N hydrochloric acid and 35 ml of ethyl acetate, and the phases areseparated. The ethyl acetate phase is back-extracted once with 30 ml of1N hydrochloric acid. The combined aqueous phases are extracted threetimes with 10 ml of diethyl ether each time. The aqueous phase is madealkaline with NaOH and extracted with ethyl acetate. The organic phasesare dried over sodium sulfate and concentrated.

7.4 g (80%) of product are obtained

Melting point: 126° C.

¹H-NMR (DMSO-D₆): 2.4 (s, 3H) ppm, 3.5 (s, 3H) ppm, 6.0 (s, 1H) ppm, 7.2(m, 1H) ppm, 7.4 (m, 2H) ppm, 8.0 (m, 1H) ppm, 8.55 (d, J=2 Hz, 1H) ppm,9.75 (s, NH) ppm.

The (−)-enantiomer is obtained after separation of the enantiomers onchiral columns (Chiralpak AS from Baker, mobile phasen-heptane/ethanol=8:2).

Melting pont: 117° C. (from ethanol)

[α]_(D) ²⁰: −62.8° (methanol)

Example XIII Methyl(R)-6-bromomethyl-4-(2-chloro-4-fluorophenyl)-2-(3,5-difluoro-2-pyridyl)-1,4-dihydropyrimidine-5-carboxylate

A solution of 2 g (5.05 mmol) of methyl4-(2-chloro-4-fluorophenyl)-2-(3,5-difluoropyridin-2-yl)-6-methyl-1,4-dihydropyrimidine-5-carboxylatefrom Example XII in 30 ml of tetrachloromethane is heated to 50° C.under argon, resulting in a clear solution. At this temperature, 0.99 g(5.56 mmol) of N-bromosuccinimide is added, and the mixture is kept atthis temperature for 10 minutes. It is immediately cooled, filtered withsuction and concentrated under reduced pressure at room temperature. Theproduct is more than 90% pure according to HPLC and is employed furtheras crude material.

R_(f)=0.33 (cyclohexane/ethyl acetate=7:3)

The following were prepared analogously:

Example Methyl 6-bromomethyl-4-(2,4-dichlorophenyl)-2-(3,5- XIV:difluoro-2-pyridyl)-1,4-dihydropyrimidine-5-carboxylate Example Methyl6-bromomethyl-4-(2-chlorophenyl)-2-(3,5-difluoro-2- XV:pyridyl)-1,4-dihydropyrimidine-5-carboxylate Example Methyl6-bromomethyl-4-(2,4-difluorophenyl)-2-(3,5- XVI:difluoro-2-pyridyl)-1,4-dihydropyrimidine-5-carboxylate Example Methyl6-bromomethyl-4-(2-chloro-4-fluorophenyl)-2-(2- XVII:thiazolyl)-1,4-dihydropyrimidine-5-carboxylate Example Methyl6-bromomethyl-4-(2-bromo-4-fluorophenyl)-2-(2 XVIII:thiazolyl)-1,4-dihydropyrimidine-5-carboxylate

B. Preparation Examples Example 1 Methyl4-(2-chloro-4-fluorophenyl)-2-(3,5-difluoro-2-pyridyl)-6-(4-morpholinylmethyl)-1,4-dihydropyrimidine-5-carboxylate

A solution of 0.38 g (2.4 mmol) of 2-chloro-4-fluorobenzaldehyde in 10ml of isopropanol is heated together with 0.46 g (2.4 mmol) of thecompound from Example VI, 0.48 g (2.4 mmol) of the compound from ExampleVIII and 0.24 g (2.88 mmol) of sodium acetate under reflux for 2 hours.The reaction mixture is concentrated, and the residue is taken up indichloromethane and extracted with 2N hydrochloric acid. The aqueousphase is made alkaline with dilute ammonia solution and extracted withdichloromethane. The organic phase is washed with aqueous sodiumchloride solution, dried over sodium sulfate and concentrated. Theresidue is chromatographed on silica gel with cyclohexane/ethyl acetate(20:1→10:1) as mobile phase, and the product is crystallized fromdiethyl ether.

Yield: 0.03 g (2.6%)

Melting point: 190° C.

Example 2 Methyl4-(2-chloro-4-fluorophenyl)-2-(3,5-difluoro-2-pyridyl)-6-[2-(1,3-thiazolidin-3-yl)-ethyl]-1,4-dihydropyrimidine-5-carboxylate

A solution of 0.20 g (0.56 mmol) of the compound from Example IX in 5 mlof isopropanol is heated together with 0.11 g (0.56 mmol) of thecompound from Example VI and 0.06 g (0.67 mmol) of sodium acetate underreflux for 2 hours. The reaction mixture is concentrated, and theresidue is taken up in ethyl acetate and extracted with dilutehydrochloric acid. The aqueous phase is made alkaline with dilute sodiumhydroxide solution and extracted with ethyl acetate. The organic phaseis dried over sodium sulfate and concentrated. The residue ischromatographed on silica gel with cyclohexane/ethyl acetate (5:1→3:1)as mobile phase.

Yield: 0.028 g (10.1%)

Melting point: 130° C.

Example 3 Methyl4-(2-chloro-4-fluorophenyl)-2-(3-fluoro-2-pyridinyl)-6-[2-(4-pyridinyl)-ethyl]-1,4-dihydropyrimidine-5-carboxylate

0.60 g (1.73 mmol) of the benzylidene compound from Example XI and 0.30g (1.73 mmol) of the compound from Example III are heated together with0.17 g (2.08 mmol) of sodium acetate in 12 ml of isopropanol underreflux overnight. The mixture is concentrated, taken up indichloromethane and extracted with 2N hydrochloric acid. The aqueousphase is made alkaline with sodium hydroxide solution and extracted withdichloromethane, and the organic phase is washed with aqueous sodiumchloride solution, dried over sodium sulfate and concentrated. Theresidue is chromatographed on silica gel with cyclohexane/ethyl acetate(1:1) as mobile phase, and further purified by crystallization fromdiethyl ether.

Yield: 0.04 g (5% of theory)

R_(f)=0.79 (dichloromethane/methanol 10:1)

Example 4 Methyl(R)-4-(2-chloro-4-fluorophenyl)-2-(3,5-difluoro-2-pyridyl)-6-piperidinomethyl-1,4-dihydropyrimidine-5-carboxylate

A solution of 100 mg of freshly prepared methyl(R)-6-bromomethyl-4-(2-chloro-4-fluorophenyl)-2-(3,5-difluoro-2-pyridyl)-1,4-dihydropyrimidine-5-carboxylatefrom Example XIII in 0.5 ml of methanol is mixed with 5 equivalents ofpiperidine and stirred at room temperature for 30 minutes. The solutionis diluted with water and extracted with ethyl acetate.

Yield: 78 mg

Melting point: 132° C.

The compounds listed in the following table were prepared in analogousmanner.

Mobile Ex. phase No. m.p. (°C.) R_(f) system 5

178 6

0.46 B 7

182–183 8

155–156 9

216–218 10

161–162 11

0.34 C 12

225–227 13

168–170 14

189–191 15

166–168 16

142–143 17

0.13 E 18

128–131 19

175 20

137 21

172–173 22

0.29 F 23

0.20 F 24

156–158 25

0.60 G 26

0.07 C 27

155 28

183–184 29

173–174 30

144–145 31

189–190 32

114–116 33

0.54 E 34

161–163 35

117–119 36

145 37

163–164 38

119–120 39

— 0.13 H 40

— 0.12 I 41

121 42

135 43

128 44

0.51 E 45

149 46

169–170 47

133 48

132 49

0.49 E 50

0.21 F 51

0.23 F 52

123–126 53

0.18 C 54

0.44 C

Mobile phase Abbreviation Cyclohexane/ethyl acetate = 7:3 ACyclohexane/ethyl acetate = 8:2 B Cyclohexane/ethyl acetate = 1:1 CCyclohexane/ethyl acetate = 7:3 + D drops of NH₃ Methylenechloride/methanol = 95:5 E Methylene chloride/methanol = 9:1 FToluene/acetone = 1:1 G Toluene/methanol 10:1 H Toluene/ethyl acetate =4:1 I Methylene chloride/methanol = 10:1 J Methylene chloride/methanol =95:5 + K drops of NH₃The activity data for some compounds of the invention are listed below:

Example No. IC₅₀ (μM) Tox₅₀ (μM) 2 0.4 38 5 0.002 40 7 0.025 25 9 0.00717 11 0.04 >8 15 0.05 3 24 0.02 80 31 0.002 63 34 0.009 60 45 0.002 34The treatment of the hepatitis B virus-producing HepG2.2.15 cells withthe compounds of the invention surprisingly led to a reduction in intra-and/or extracellular viral DNA.

The invention claimed is:
 1. A compound of the formula

and the isomeric form thereof

in which R¹, R² are, independently of one another, hydrogen, fluorine,chlorine or bromine, R³ is C₁–C₄-alkyl, X is a methylene or ethylenegroup, Z is NR⁴R⁵ or pyridyl, R⁴ is C₁–C₄-alkyl which may be substitutedby hydroxyl or C₁–C₄-alkoxycarbonyl, or is benzyl, R⁵ is C₁–C₄-alkylwhich may be substituted by hydroxyl, or R⁴ and R⁵ together with thenitrogen atom to which they are bonded are an imidazolyl, triazolyl ortetrazolyl ring or a radical of the formula

in which a is zero or 1 and Y is CH₂, CH₂CH₂, —O— or —S—, and R₆ ispyridyl which is substituted once to twice by fluorine, or is thiazolyl,and the salts thereof.
 2. A compound as claimed in claim 1, in which R⁴and R⁵ together with the nitrogen atom to which they are bonded form amorpholinyl or thiomorpholinyl ring, and the salts thereof.
 3. Acompound as claimed in claim 1, in which R¹, R² are, independently ofone another, fluorine, chlorine or bromine, and the salts thereof.
 4. Acompound as claimed in claim 1, in which R¹, R² are, independently ofone another, fluorine, chlorine or bromine, and R⁴ and R⁵ together withthe nitrogen atom to which they are bonded form a morpholinyl orthiomorpholinyl ring, and the salts thereof.
 5. A compound selected fromthe group consisting of:


6. A process for preparing the compounds as claimed in claim 1, by [A]reacting compounds of the formula

in which R¹ to R³, X and Z have the meanings indicated in claim 1, withamidines of the formula

in which R⁶ has the meaning indicated in claim 1, or the salts thereof,or [B] reacting compounds of the formula

in which R³, X and Z have the meanings indicated in claim 1, in aone-stage process with aldehydes of the formula

in which R¹ and R² have the meanings indicated in claim 1, and amidinesof the formula

in which R⁶ has the meanings indicated in claim 1, or the salts thereof,or else [C] where X in formula (I) is a methylene group, reactingcompounds of the formula

in which R¹ to R³ and R⁶ have the meanings indicated in claim 1, and Yis a nucleophilically replaceable group, with compounds of the formula

in which R⁴ and R⁵ have the meanings indicated claim 1, or else [D]where X in formula (I) is an ethylene group, reacting compounds of theformula

in which R¹ to R⁵ have the meanings indicated in claim 1, with amidinesof the formula

in which R⁶ has the meanings indicated in claim 1, or the salts thereof.7. A process for preparing the compounds as claimed in claim 1, by [A]firstly converting aldehydes of the formula

in which R¹ and R² have the meanings indicated in claim 1, with β-ketoesters of the formula

in which R³, X and Z have the meanings indicated in claim 1, intobenzylidene compounds of the formula

and then reacting the latter with amidines of the formula

in which R⁶ has the meaning indicated in claim 1, or the salts thereof,or [B] reacting compounds of the formula (III) in a one-stage processwith aldehydes (II) and amidines (V) or the salts thereof, or else [C]where X in formula (I) is a methylene group, reacting compounds of theformula

in which R¹ to R³ and R⁶ have the meanings indicated in claim 1, and Yis a nucleophilically replaceable group, with compounds of the formula

in which R⁴ and R⁴ have the meanings indicated in claim 1, or else [D]where X in formula (I) is an ethylene group, converting compounds of theformula

in which R¹ to R³ have the meanings indicated in claim 1, with immoniumsalts of the formula

in which R⁴ and R⁵ have the meanings indicated in claim 1, intocompounds of the formula

and then reacting the latter with amidines of the formula (V) or thesalts thereof.
 8. A compound of the formula

in which R¹, R², R³ and R⁶ have the meanings indicated in claim
 1. 9. Amethod of treating hepatitis B infection, comprising administering to amammal an effective amount of a compound of claim
 1. 10. A method oftreating diseases caused by infection with hepatitis B virus, comprisingadministering to a mammal an effective amount of a compound of claim 1.11. The method of claim 10 wherein said disease is hepatitis.
 12. Themethod of claim 10 wherein said disease is cirrhosis of the liver. 13.The method of claim 10 wherein said disease is hepatocellular carcinoma.14. A pharmaceutical preparation comprising one or more compounds ofclaim 1 in combination with a pharmaceutically acceptable carrier.